Contribution to the structural characterization of eucaryotic PSI reaction centre — II. Characterization of a highly purified photoactive SDS-CP1 complex

Under precise conditions, SDS PAGE allows purification of a photoactive P700-chla-protein complex from eucaryotic cells. The yield of P700 recovery is close to 100%. A total protein content equivalent to about 140 kD for one mole of P700 has been estimated by chemical analysis, and electrophoresis revealed the presence of two peptidic chains with MWs close to 65 kD. Photochemical and structural properties of this complex are given and compared with those of other complexes previously isolated.     

Evolution of isozyme loci and their differential tissue expression

Summary The phylogeny of the creatine kinase (CK, EC 2.7.3.2) isozyme loci and their differential tissue expressions were determined for representatives of 65 families of vertebrates, with emphasis on the fishes. The transition from the single creatine kinase locus, characteristic of certain echinoderms, to the two creatine kinase loci which are orthologous to those present in all vertebrates, occurred early in the chordate line. The majority of pre-teleostean fishes possesses only these two CK loci (A and C). These loci are relatively generalized in their tissue expressions which are variable among species of primitive fishes. The third and fourth creatine kinase loci (B and D) arose separately in the ancestors of the bony fishes and appear to be the result of regional genome duplications. Concomitant with the increase in the number of isozyme loci has been an increase in the specificity of their tissue expression. In the advanced teleost fishes the four CK loci are differentially expressed in a characteristic manner. The A₂ isozyme predominates in skeletal muscle, the B₂ isozyme in eye and brain, the C₂ isozyme in stomach muscle, and the D₂ isozyme is found exclusively in testis. We propose a phylogeny of the creatine kinase genes in the lower chordates based on the time of appearance of new CK loci, the sequence in which the loci achieve a tissue restricted expression, and the immunochemical relatedness of the orthologous and paralogous gene products.     

Controlling vitrification of petunia in vitro

Summary Nodal segments from in vitro culturedPetunia hybrida were grown under varying cultural conditions. The origin of nodal explants influenced vitrification. Basal segments formed a higher percentage of vitreous shoots than did the upper nodes. A method was developed for including polyethylene glycol with Gelrite to obtain gelled media of varying water potentials. Media water potential from −0.31 to −1.2 MPa had no effect on controlling the level of vitrification. Gelrite promoted vitrification but GIBCO agar, alone or in combination with Gelrite, reduced its occurrence. Lowering media NH₄ content reduced vitrification, whereas sealing culture vessels with parafilm increased it. As it is now possible to control normal and vitreous plant development inPetunia, this can be used as a model system for studying the physiology and biochemistry of this developmental abnormality.     

A survey of seasonal bark proteins in eight temperate hardwoods

Summary Bark proteins of eight temperate hardwoods were analyzed by SDS-PAGE at monthly intervals to determine whether an accumulation of specific proteins, potential storage proteins, occurred in the fall at the time of leaf senescence. Storage proteins were identified as proteins that accumulated during the fall and were present in reduced amounts in the summer. Total protein levels were higher in the winter than in the summer in Fagus sylvatica, Fraxinus americana, Tilia americana, Alnus glulinosa, Betula papyrifera and Querus rubra, but not in Gleditsia triacanthos or Robinia pseudoacacia. Betula contained the most abundant storage protein, although in all species minor bands, which fluctuated seasonally, could be identified. With the exception of Alnus and Betula, results generally correlated with previous microscopy studies of these tree species, which showed varying amounts of protein storage vacuoles present in phloem parenchyma cells during the winter, but not during the summer.     

Ammonium chloride inhibits basal degradation of newly synthesized collagen in human fetal lung fibroblasts

The objective of this work was to characterize basal degradation of newly synthesized collagen in human fetal lung fibroblasts. Analysis of 22 separate determinations showed that in cells incubated under normal conditions, the level of intracellular degradation was normally distributed with a mean of 15.2% and a standard deviation of 2.6%. Within each experiment, however, the uncertainty (standard deviation) in determining degradation was very small, usually less than 1.5%. Consideration of the large variation between experiments and the ability of our analytic technique to detect small, but "statistically significant," differences between groups within the same experiment led us to formulate two criteria for determining whether degradation measured in cultures exposed to some agent differs in a "biologically significant" way from degradation measured in control cultures. These criteria were used to evaluate the effects of the following proteinase inhibitors on basal degradation: NH4Cl, which increases the pH of subcellular compartments that are normally acidic; and leupeptin and Na-p-tosyl-L-lysine chloromethyl ketone (TLCK), which are inhibitors of lysosomal cathepsins (B and L) that degrade collagen. NH4Cl (16 mM) lowered degradation to an extent that was both statistically and biologically significant, but neither leupeptin nor TLCK affected degradation. The effect of NH4Cl on degradation was independent of its inhibitory effects on production of collagen, protein, and ATP. These results suggest that basal degradation occurs in, or beyond, an acidic (i.e., NH4Cl-sensitive) but nonlysosomal compartment of the cell, and that NH4Cl inhibits processing within, or transport to, that compartment. This is the first report of an agent that inhibits basal degradation of newly synthesized collagen in soft tissue fibroblasts.     

Collagen degradation in I-cells is normal

There is evidence that lysosomal proteases mediate the intracellular degradation of structurally abnormal collagen. I-Cell disease (Mucolipidosis II) is characterized by marked deficiency of many lysosomal hydrolases, including the collagenolytic enzyme cathepsin B. The experiments reported here tested the hypothesis that degradation of abnormal collagen would be severely impaired in I-cells. Skin fibroblasts from 3 patients with I-cell disease were incubated with and without cis-hydroxyproline, a proline analog that causes structural abnormalities in collagen, and [14C]proline. The amount of [14C]hydroxyproline in a low molecular weight fraction relative to total [14C]hydroxyproline was used as a measure of intracellular collagen degradation. Levels of degradation were significantly higher in I-cells exposed to cis-hydroxyproline than in cells incubated without the analog. Similar data were obtained for normal human fetal lung fibroblasts incubated under the same conditions. Degradation of [125I]-epidermal growth factor was used to assess the functionality of the lysosomal pathway for protein degradation, and it was much lower in I-cells than in normal cells. It can be concluded that a completely functional complement of lysosomal enzymes is not necessary for structurally abnormal collagen to be degraded intracellularly; the data suggest that a nonlysosomal pathway exists.     

The genetic defect in multiple endocrine neoplasia type 2A maps next to the centromere of chromosome 10

Multiple endocrine neoplasia type 2A (MEN2A) is a rare cancer syndrome that is inherited in an apparently autosomal dominant fashion. Previous linkage studies had assigned the MEN2A locus to chromosome 10 in the pericentromeric region. We recently have described several new easily scorable RFLPs for the chromosome 10-specific alpha satellite DNA (the D10Z1) locus that is known, on the basis of previous in situ hybridization experiments, to lie at the centromere. We report here tight linkage between MEN2A and D10Z1, as demonstrated by a maximum lod score of 12.02 at the recombination frequency of zero (1-lod-unit support interval 0-4 cM), indicating that the genetic defect in MEN2A lies in the immediate vicinity of the centromere. By means of a set of ordered polymorphic DNA markers from the pericentromeric region, multipoint as well as pairwise linkage analyses place the MEN2A locus at the middle of a small region (approximately 11 cM) bracketing the centromere with FNRB (at 10p11.2) and RBP3 (at 10q11.2) on either side, providing further support for the centromeric location of the MEN2A locus. Marked sex difference in recombination frequencies exists in this pericentromeric region: significantly (P less than .01) more female than male crossovers were observed across all of the adjacent intervals D10S24-FNRB, FNRB-D10Z1, and D10Z1-RBP3. However, a sex difference was not seen in the 7-cM interval from RBP3 to D10S5, suggesting that large variation in the sex difference in recombination can occur over small chromosomal regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

The human ryanodine receptor gene: Its mapping to 19q13.1, placement in a chromosome 19 linkage group, and exclusion as the gene causing myotonic dystrophy

The recent cloning of cDNA encoding the Ca++ release channel (ryanodine receptor) of human sarcoplasmic reticulum has enabled us to use somatic cell hybrids to localize the ryanodine receptor gene (RYR) to the proximal long arm of human chromosome 19. Studies with additional hybrids containing deletions or translocations in chromosome 19 enabled us to localize RYR to 19q13.1 in a region distal to GPI/MAG and proximal to D19S18/DNF11. On the basis that the myotonic dystrophy (DM) locus maps near this region and that myotonia could result from a defect in the ryanodine receptor, we examined the linkage between the DM locus and RYR. Our results, showing several DM-RYR recombinants, rule out an RYR defect as the cause of DM. However, localization of RYR to a region of human chromosome 19 which is syntenic to an area of pig chromosome 6 containing the HAL gene responsible for porcine malignant hyperthermia supports the candidacy of RYR for this disorder.     

Effect of angiotensin II and norepinephrine on release of prostaglandins E2 and I2 by the perfused rat mesenteric artery

Prostaglandin E₂ (PGE₂) and 6 keto-PGF_1α, the stable metabolite of prostacyclin (PGI₂), have been measured in the effluent of perfused rat mesenteric arteries by the use of a sensitive and specific radioimmunoadday (RIA) method. The PGE₂ and 6-keto-PGF_1α were continuousyl released by the unstimulated mesenteric artery over a period of 145 min. After 100 min of perfusion the release of PGE₂ and 6-keto-PGF_1α was 4.5 ± 8.4 pg/min and 254 ± 75 pg.min respectively, which is in accord with the general belief that PGI₂ is the major PG synthesized by arterial tissue. Angiotensin II (AII) 5 ng/ml) induced an increased of PGE₂ and 6-keto-PGF_1α release without changing the perfusion pressure. The effect of norepinephrine (NE) injections on release of PGs depended on the duration of the stabilization period. The changes of perfusion pressure induced by NE were not related to changes in release of PGs. Thus, it seems that the increase of PG release induced by AII and NE was due to a direct effect of the drugs on the vascular wall. This may represent an important modulating mechanism in the regulation of vascular tone.